Stoichiometry of the proton-ATPase of growing and resting, aerobic Escherichia coli

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Abstract
The H+/ATP stoichiometry of the proton-translocating ATPase was investigated in growing and nongrowing, respiring cells of E. coli. The protonmotive force, .DELTA.p, was determined by measuring the transmembrane chemical gradient of protons, .DELTA.pH, from the cellular accumulation of benzoate anions, and the electrical gradient, .DELTA..PSI., from the accumulation of the lipophilic cation tetraphenylphosphonium (TPP+). The accumulation of lactose was also used to calculate the .DELTA.p in this lactose operon constitutive, .beta.-galactosidase negative mutant. The phosphorylation potential, .DELTA.Gp'', was determined by measuring the cellular concentration of ATP, ADP and Pi. According to chemiosmotic principles, at steady state the phoshorylation potential is in thermodynamic equilibrium with the protonmotive force, and thus the ratio .DELTA.p/.DELTA.Gp'' can be used to determine the H+/ATP ratio. Respiring E. coli cells, in mid-exponential phase of growth or incubated in buffer, at external pH from 6.25-8.25 had a constant .DELTA.Gp'' of .apprx. 500 mV. The H+/ATP ratio was 3 when the .DELTA.p value derived from lactose accumulation levels was used. When the .DELTA.p values derived from .DELTA.pH and .DELTA..PSI. were used in the calculations, the H+/ATP ratio varied from .apprx. 2.5 at external pH 6.25 to .apprx. 4 at pH 8.25. Arguments are presented for the hypothesis that the .DELTA..PSI. values obtained from the TPP+ measurements are likely to be inaccurate and that a value of 3 H+/ATP, independent of the external pH, is likely to be the valid stoichiometry.